In vitro and in silico studies on fibrinolytic activity of nattokinase: A clot buster from Bacillus sp.

doi:10.1007/s11515-017-1453-3

Front. Biol.

2017, Vol. 12

Issue (3) : 219-225 https://doi.org/10.1007/s11515-017-1453-3

RESEARCH ARTICLE

In vitro and in silico studies on fibrinolytic activity of nattokinase: A clot buster from Bacillus sp.

V. Mohanasrinivasan., A. Mohanapriya, Swaroop Potdar, Sourav Chatterji, Srinath Konne, Sweta Kumari, S. Merlyn Keziah., C. Subathra Devi(

)

School of Biosciences and Technology, VIT University, Vellore-632014, Tamil Nadu, India

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Abstract

BACKGROUND: Nattokinase (NK) is a serine protease enzyme of the subtilisin family. It exhibits a strong fibrinolytic activity. The fibrinolytic enzymes from? Bacillus ?sp. have attracted interest as thrombolytic agents because of their efficiency in the fibrinolytic process including plasmin activation.

METHODS: In the present study, VIT garden soil was collected and subjected to isolation process in order to screen for the NK production. Screening for NK enzyme was performed by radial caseinolytic assay. The production of NK enzyme was done in two different production medium for comparative studies. The NK enzyme was purified by gel permeation chromatography. The activity of the purified NK was checked by clot lysis and casein digestion assay. To investigate the structural basis of NK and fibrinogen interaction and also to identify the best binding mode, molecular dynamics and docking studies were performed.

RESULTS: Based on the morphological and biochemical characterization, the isolate was identified as Bacillus sp. The overall purification fold of NK was about 3 with the specific activity of 664U/mg and 9.9% yield. Homogeneity of the purified enzyme was analyzed and confirmed by the single band obtained in SDS-PAGE. Molecular weight of the purified protease was estimated as 25 kDa. Purified NK enzyme exhibited 97% of effective clot lysis activity. The NK was docked in to the knob region of the fibrinogen at its binding site using Dock server. A total of 26 residues of fibrinogen and 29 residues of NK constitute the interface region. However, 9 residues of fibrinogen (THR238, MET264, LYS266, ARG275, THR277, ALA279, ASN308, MET310, and LYS321) and 8 residues of NK (GLY61, SER63, THR99, PHE189, LEU209, TYR217, ASN218, and MET222) are involved in intact binding.

CONCLUSIONS: A significant amount of NK enzyme was obtained from Bacillus sp. The docking analysis revealed that the NK and fibrinogen adopt an extended binding pattern and interacts with the crucial residues to exhibit their activity.

Keywords nattokinase (NK) Bacillussp. clot busters docking

Corresponding Author(s): C. Subathra Devi

Online First Date: 07 June 2017 Issue Date: 19 June 2017

Cite this article:

V. Mohanasrinivasan.,A. Mohanapriya,Swaroop Potdar, et al. In vitro and in silico studies on fibrinolytic activity of nattokinase: A clot buster from Bacillus sp.[J]. Front. Biol., 2017, 12(3): 219-225.

URL:

https://academic.hep.com.cn/fib/EN/10.1007/s11515-017-1453-3
https://academic.hep.com.cn/fib/EN/Y2017/V12/I3/219

Fig.1 Bacillus sp. (A) Pure culture (B) Microscopy 40×.

Fig.2 (A) Bacillus sp. showing zone of hydrolysis on casein plate. (B) Hydrolysis of casein by Bacillus sp. expressed in mm.

Fig.3 Elution profile of NK enzyme extracted from Bacillus sp.

Fig.4 Clot lysis activity.

Fig.5 SDS-PAGE of NK enzyme.

Fig.6 Structure of NK (4DWW) visualized using Pymol.

Fig.7 Potential energy plot showing energy minimization (using GROMACS).

Fig.8 Molecular dynamics simulation results. (A) RMSD graph – RMSD of structure is within 0.2 nm suggesting the stability of NK structure. Structure reaches stable conformation after 30 ns during simulation. (B) RMSF plot – Most of the residues (other than residues at position 53, 79, 100, 160, 205, 212) have fluctuaion less than 0.2 nm which confirms the stable conformation of the NK structure. (C) Presence of hydrogen bond in each atom of NK is shown in hydrogen bond existence map. Here red and white color represents presence and absence of hydrogen bond respectively. (D) Number of hydrogen bonds within protein structure is shown in the graph. (D) Negligible variation in total energy was observed during the simulation study. Constant energy graph represents the stabilty of the structure.

Fig.9 NK docked to fibrinogen knob at its binding site using Haddock server.

Tab.1 NK enzyme activity

Tab.2 The Haddock score and energy values of NK-fibrinogen complex

Tab.3 Interaction analysis of NK-fibrinogen complex representing the interface and interacting residues

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